An atlas of sarbecovirus infectivity across diverse ACE2 orthologs

Sarbecoviruses pose a substantial zoonotic threat, yet the determinants governing their host range remain incompletely defined. To systematically investigate host entry mechanisms, we generated a panel of vesicular stomatitis virus (VSV) pseudoviruses representing 46 sarbecoviruses and assessed their infectivity across 66 angiotensin-converting enzyme 2 (ACE2) orthologs. This analysis produced a high-resolution host-entry landscape with direct implications for cross-species transmission risk. We found that all severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) isolates, WIV1, and pangolin-derived sarbecoviruses demonstrated broad ACE2 usage, whereas no sarbecovirus utilized alternative entry factors including neuropilin-1, CD147, AXL, or TMEM106B. Notably, ACE2 orthologs from Rhinolophus affinis and Miniopterus schreibersii supported infection by nearly all clade 1 sarbecoviruses, identifying highly permissive host receptors. We also identified residues 27 and 42 of ACE2 as critical determinants of sarbecovirus host tropism. Together, these findings define host entry constraints relevant to receptor-based risk assessment and inform surveillance and pandemic preparedness.